Selectivity of Protein Interactions along the Aggregation Pathway of α-Synuclein

Written by: Adriaan von dem Borne, Sophie Klouwer & Stijn Sijbesma

Background 

α-synuclein is a protein that is abundantly expressed in the brain. Aggregation of this protein is related to the formation of Lewy Bodies (LBs), which are intracellular inclusions in which α-synuclein is the main component. It is known that certain disease-associated mutations within α-synuclein can increase its propensity to aggregate. LBs are found in the brains of patients with different types of neurodegenerative diseases such as Parkinson Disease (PD), Dementia with Lewy bodies (DLB) and in some cases of Alzheimer’s disease (AD).

LBs can contain over 70 different proteins (1). What is unknown, however, is how these interact with α-synuclein. In order to understand how LBs are formed, it is crucial to know in which order and in what form LB proteins aggregate with α-synuclein. In this study, the researchers investigate the binding of LB proteins and α-synuclein in its different forms along the aggregation pathway: monomers, oligomers and finally fibrils.

 

Why this preprint is interesting

We harboured a shared personal interest in LBs and PD ever since we began our investigation in the field of protein folding and assembly. Worldwide, over 10 million people are living with PD. It is through characterization of the molecular background that advances in medical sciences to treat these PD patients can ultimately be achieved. The paper describes a novel approach to understanding critical protein interactions with α-synuclein as a supramolecular complex, which may contribute to the formation of LBs. Characterizing the interactome of α-synuclein for a more holistic understanding of LB formation could fundamentally change the way in which the aforementioned neurodegenerative diseases are understood.

 

Key findings

AlphaScreen on monomeric α-synuclein

The researchers start by studying the interactions of LB proteins with the monomeric form of α-synuclein. They do this using the ‘AlphaScreen’ method they previously developed (2). AlphaScreen is a highly sensitive proximity assay in which proteins are attached to donor and acceptor beads. When the interacting proteins get in close proximity, the donor transfers an excited oxygen atom to the acceptor. Because of this transfer, light will be emitted which can be detected. The researchers perform the AlphaScreen assay using mCherry-tagged α-synuclein and GFP-tagged LB proteins, which are co-expressed in a cell-free expression system. Therefore, the proteins are co-translated and can thus also interact during their folding processes. The researchers find three main interacting LB proteins from the AlphaScreen assay. These are: G-protein receptor kinase 5 (GRK5), mitogen-activated protein kinase 1 (MAPK1) and proteasome activator complex subunit 1 (PSME1). This relatively small number of hits of only three interacting proteins can be explained by the fact that other LB proteins may bind to other (non-monomeric) forms of α-synuclein.

 

Single molecule fluorescence: selective recognition of Lewy Body proteins by α-synuclein oligomers

The authors then detect ‘co-assembly events’ of GFP-tagged LB proteins and mCherry-tagged α-synuclein by Two-Color Coincidence Detection (TCCD) with pathological α-synuclein mutants A30P, G51D and A53T in nanotraps. These particular mutants are chosen because of their known ability to form oligomers (3). This approach results in the observation of similar interactomes for the three mutants, and reveals that the binding to oligomeric α-synuclein occurs in sub-stoichiometric ratios. However, the small heat shock protein αB-crystallin is present in complexes of higher stoichiometry with α-synuclein. The tendency of αB-crystallin to self-aggregate may explain this result (4).

 

Co-aggregation or recognition of pre-formed α-synuclein oligomers? 

The binding partners of α-synuclein can either bind through co-aggregation during oligomer formation, or bind through recognition of fully formed α-synuclein oligomers. In the human brain, α-synuclein oligomers will form in the presence of interacting proteins. Therefore, co-aggregation processes may play an important role. To discriminate between the two options, the authors add possible interacting proteins during α-synuclein oligomerization and after formation of these aggregate species.

The authors see that most interacting proteins do not have the ability to recognize pre-formed aggregates, meaning that recognition of α-synuclein happens during oligomerization. However, proteins from the LC3/GABARA family do recognize pre-formed α-synuclein oligomers. These proteins are involved in autophagy suggesting that this interaction may be relevant for the degradation of pathological α-synuclein.

 

Monitoring interacting proteins of α-synuclein fibrils 

To determine the interactome of the α-synuclein fibrils, the researchers use fluorescently labelled pre-formed fibrils and add them to the possible binding partners. The authors find a high number of binding partners, including many proteins that did not co-diffuse with α-synuclein oligomers. The Tau protein is one of these interactors. In some patients suffering from neurodegenerative diseases, both α-synuclein and Tau aggregates are present. A possible connection may be that Tau monomers bind to the surface of α-synuclein fibrils, inducing nucleation of Tau aggregation (5).

The authors find that different chaperones interact with different α-synuclein aggregation states. Whereas the small heat shock protein αB-crystallin recognizes early misfolded states, Hsp40 chaperones recognize the fibrillar stages. These interactions explain why chaperones are universally present in brain inclusions like LBs.

In summary, this preprint demonstrates for the first time on a large scale that interacting proteins recognize different conformers of α-synuclein along the aggregation pathway from monomer to fibril, illuminating the pathogenic process of LB formation.

 

Questions

• Do you think that the point mutations could also affect binding to α-synuclein monomers as detected in the AlphaScreen assay?
• Did you observe any fibril formation after a long incubation time of the α-synuclein oligomers in the cell-free expression system? Do you think the formation of fibrils in the presence of the possible interactors may generate different results than the pre-formed fibrils did?
• Could you elaborate on why TCCD is a good method for the experiments conducted in this paper? For example how does TCCD compare to split-GFP experiments?
• Do you expect there to be common characteristics among the interacting proteins having a preference for a certain folding state of α-synuclein?

 

References

1. Wakabayashi, K., Tanji, K., Mori, F., & Takahashi, H. (2007). The Lewy body in Parkinson’s disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates. Neuropathology : official journal of the Japanese Society of Neuropathology, 27(5), 494–506. https://doi.org/10.1111/j.1440-1789.2007.00803.x.
2. Sierecki, E., Stevers, L. M., Giles, N., Polinkovsky, M. E., Moustaqil, M., Mureev, S., Johnston, W. A., Dahmer-Heath, M., Skalamera, D., Gonda, T. J., Gabrielli, B., Collins, B. M., Alexandrov, K., & Gambin, Y. (2014). Rapid Mapping of Interactions between Human SNX-BAR Proteins Measured In Vitro by AlphaScreen and Single-molecule Spectroscopy. Molecular & Cellular Proteomics, 13(9), 2233–2245. https://doi.org/10.1074/mcp.m113.037275.
3. Sierecki, E., Giles, N., Bowden, Q., Polinkovsky, M.E., Steinbeck, J., Arrioti, N., Rahman, D., Bhumkar, A., Nicovich P.R., Ross, I., Parton, R.G., Böcking, T. & Gambin, Y (2016). Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence. Scientific Reports 6, 37630. https://doi.org/10.1038/srep37630.
4. Hilton, G. R., Hochberg, G. K., Laganowsky, A., McGinnigle, S. I., Baldwin, A. J., & Benesch, J. L. (2013). C-terminal interactions mediate the quaternary dynamics of αB-crystallin. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 368(1617), 20110405. https://doi.org/10.1098/rstb.2011.0405.
5. Giasson, B. I., Forman, M. S., Makoto Higuchi, M., Golbe, L.I., Graves, C. L., Kotzbauer, P. T., Trojanowski, J. Q., Lee, V. M. Y. (2003). Initiation and Synergistic Fibrillization of Tau and Alpha-Synuclein. Science, 300(5619) pp. 636-640
   https://doi.org/10.1126/science.1082324.

Tags: alphascreen, lewy bodies, protein-protein interactions, two-color coincidence detection, α-synuclein

doi: Pending

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